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A fluorescence microscope uses fluorescent chromophores called fluorochromes, which can absorb energy from a light source and then emit this energy as visible light. Fluorochromes include naturally fluorescent substances (such as chlorophylls) and fluorescent stains that are added to the specimen to create contrast. Dyes such as Texas red and FITC are examples of fluorochromes. Other examples include the nucleic acid dyes 4’,6’-diamidino-2-phenylindole (DAPI), and acridine orange.

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Updated: Jun 26, 2025

A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

Published on: March 30, 2020

Precise immunofluorescence canceling for highly multiplexed imaging to capture specific cell states.

Kosuke Tomimatsu¹, Takeru Fujii¹, Ryoma Bise²

¹Division of Transcriptomics, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-0054, Japan.

Nature Communications

|May 8, 2024

Summary

Precise Emission Canceling Antibodies (PECAbs) enable high-specificity sequential imaging for analyzing cell signaling dynamics. This novel method allows for detailed reconstruction of spatiotemporal signaling pathways and cell states in human tissue.

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Published on: April 7, 2023

Area of Science:

Cellular and Molecular Biology
Biotechnology
Immunohistochemistry

Background:

Cell states are governed by signaling pathways responding to external stimuli.
High-resolution and multiplexed imaging have advanced spatial protein analysis but face antibody specificity challenges.
Visualizing activated signaling pathways requires improved specificity in imaging techniques.

Purpose of the Study:

To develop a novel antibody-based imaging system for high-specificity visualization of cellular signaling.
To enable the reconstruction of spatiotemporal dynamics of signaling pathways.
To create a comprehensive platform for analyzing complex cellular processes and cell states.

Main Methods:

Development of Precise Emission Canceling Antibodies (PECAbs) with cleavable fluorescent labeling.
Implementation of high-specificity sequential imaging using hundreds of PECAbs.
Integration of PECAbs with sequential fluorescence in situ hybridization (seq-smFISH) for cellular classification and signal activation state identification.

Main Results:

PECAbs provide high specificity for visualizing activated signals, overcoming limitations of current methods.
The PECAb system enables high-specificity sequential imaging of hundreds of antibodies.
Reconstruction of spatiotemporal dynamics of signaling pathways is achieved.
Combined approach effectively classifies cells and identifies signal activation states in human tissue.

Conclusions:

The PECAb system offers a powerful platform for high-specificity, sequential imaging of cellular signaling.
This technology allows for detailed spatiotemporal analysis of signaling pathways.
The integration with seq-smFISH enhances cell classification and signal state identification in tissue samples.